Omega-3 Diglyceride Emulsions

ABSTRACT

The present invention relates to omega-3 diglyceride emulsions characterized in that the lipid phase comprises at least about 40 wt.-% of diglycerides. Preferably about 70 wt.-% of the acyl-groups of said diglycerides are eicoapentaenoic acid (EPA) groups and/or docasahexaenoic (DHA) groups. The invention further relates to methods of treatment using the omega-3 diglyceride emulsions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/783,779, filed Mar. 4, 2013, which is a divisional application ofU.S. application Ser. No. 12/441,795, filed Dec. 8, 2009, now U.S. Pat.No. 8,410,181, which claims priority from International PatentApplication No. PCT/US07/020364, filed Sep. 19, 2007, which claimspriority from U.S. Provisional Application Ser. No. 60/845,518 filedSep. 19, 2006. The contents of which are hereby incorporated byreference in their entirety.

INTRODUCTION

The present invention relates to omega-3 diglyceride emulsionscharacterized in that the lipid phase comprises at least about 40 wt.-%of diglycerides. Preferably about 70 wt.-% of the acyl-groups of saiddiglycerides, are eicosapentaenoic acid (EPA) groups and/ordocosahexaenoic (DHA) groups. The invention further relates to methodsof treatment using the omega-3 diglyceride emulsions.

BACKGROUND OF THE INVENTION

Typically, post-operative and post-traumatic conditions as well assevere septic episodes are characterized by a substantial stimulation ofthe immune system ischemia reperfusion syndrome, and tendency forthrombosis formation. The immune response is activated by the release ofpro-inflammatory cytokines (e.g., tumor necrosis factor andinterleukins) which, at high levels, may cause severe tissue damage.

In such clinical conditions, it is of particular importance to provideexogenous lipids that are hydrolyzed and eliminated faster thanendogenous lipids (to avoid excessive increases of plasma triglycerideconcentration). These lipids supply fatty acids omega-3 fatty acids)capable of reducing cytokine production as well as cytokine toxicity ontissues. Free fatty acids may not be directly administered through thediet or by other parenteral means because they behave as detergents andhave toxic side effects. Thus, fatty acids are administered via lipidglycerides such as triglycerides. The fatty acids are released and usedafter the lipids are catabolized in the body via lipolysis. This effectis obtained when fatty acids are cleaved from the lipid molecules andincorporated (in free form or as components of phospholipids) in cellmembranes where they influence membrane structure and cell function,serve as secondary messengers (thus affecting regulation of cellmetabolism), influence the regulation of nuclear transcription factors,and are precursors of eicosanoids. Thus, it is desirable that thisprocess take place as quickly as possible.

The human body is capable of synthesizing certain types of fatty acids.However, omega-3 and omega-6 are designated as “essential” fatty acidsbecause they cannot be produced by the human body and must be obtainedthrough other sources. For example, fish oils from cold-water fish havehigh omega-3 polyunsaturated fatty acids content with lower omega-6fatty acid content. Most vegetable oils (i.e., soybean and safflower)have high omega-6 polyunsaturated fatty acids (most in the form of 18:2(Δ^(9, 12))-linoleic acid) content but low omega-3 (predominantly 18:3(Δ^(9, 12, 15))-α-linolenic acid) content.

Essential fatty acids may be obtained through diet or other enteral orparenteral administration. However, the rate of EPA and DHA omega-3fatty acid enrichment following oral supplementation variessubstantially between different tissues and is particularly low in someregions of the brain and in the retina especially when given as theessential fatty acid precursor, α-linolenic acid. Further, humanconsumption of omega-3 fatty acids has decreased over the past thirtyyears, while consumption of omega-6 fatty acids has increased,especially in Western populations.

U.S. application Ser. No. 11/558,568, incorporated herein by referencein its entirety, refers to methods of limiting cell death resulting fromhypoxia-ischemia comprising, administering an omega-3 lipid-basedemulsion after a hypoxia-ischemia insult. The omega-3 lipid-basedemulsion preferably comprises at least 20% omega-3 oil, by weight, andwherein the omega-3 oil comprises at least 20% omega-3 triglyceridesand/or diglycerides, and wherein fatty acids of the omega-3 triglycerideand/or diglycerides comprise at least 40% EPA and/or DHA. Theapplication also refers to novel fish-oil compositions foradministration after an ischemic insult to limit cell death in the organthat underwent an ischemic event.

Cao et al., “Chronic administration of ethyl docosahexaenoate decreasesmortality and cerebral edema in ischemic gerbils”, Life Sci. 2005 Nov.19; 78(1):74-81 alleges that dietary docosahexaenoic acid (DHA) intakecan decrease the level of membrane arachidonic acid (AA), which isliberated during cerebral ischemia and implicated in the pathogenesis ofbrain damage. Cao investigated the effects of chronic ethyldocosahexaenoate (E-DHA) administration on mortality and cerebral edemainduced by transient forebrain ischemia in gerbils.

GB 2388026, incorporated herein by reference in its entirety, refers touse n-3 polyunsaturated fatty acids EPA and/or DHA in the preparation ofan oral medicament for preventing cerebral damage in patients havingsymptoms of atherosclerosis of arteries supplying the brain.

Strokin M, Neuroscience 2006 Jun. 30; 140(2):547-53, incorporated hereinby reference in its entirety, investigated the role of docosahexaenoicacid (22:6n-3) in brain phospholipids for neuronal survival.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical composition comprising:an omega-3 diglyceride emulsion suitable for administration to apatient; and a pharmaceutically acceptable carrier, wherein the emulsionhas a lipid phase of at least about 40 wt.-% of omega-3 diglycerides,and at least about 70 wt.-% of the acyl-groups of the diglyceridescomprise EPA, DHA or a mixture thereof.

The present invention also provides a method for preventing organ deathor injury comprising: administering to a patient a therapeuticallyeffective amount of a pharmaceutical composition which comprise anomega-3 diglyceride emulsion suitable for administration to a patient;and a pharmaceutically acceptable carrier, wherein the emulsioncomprises at least about 40 wt.-% of omega-3 diglycerides.

The present invention further provides a method for reducing adversecytokine production comprising administering to a patient in needthereof a therapeutically effective amount of a pharmaceuticalcomposition comprising: an omega-3 diglyceride emulsion suitable foradministration to a patient; and a pharmaceutically acceptable carrier,wherein the emulsion comprises at least about 40 wt.-% of omega-3diglycerides.

The present invention further provides a method for reducing cell deathor damage resulting from hypoxia-ischemia comprising administering to apatient in need thereof a therapeutically effective amount of apharmaceutical composition comprising: an omega-3 diglyceride emulsionsuitable for administration to a patient; and a pharmaceuticallyacceptable carrier, wherein the emulsion comprises at least about 40wt.-% of omega-3 diglycerides.

DETAILED DESCRIPTION OF THE INVENTION

Lipid generally refers to a group of natural substances which aresoluble in hydrocarbon and insoluble in water. Lipids include anyfat-soluble (hydrophobic) naturally-occurring molecules. The term ismore specifically used to refer to fatty-acids and their derivatives(including tri-, di-, and monoglycerides and phospholipids) as well asother fat-soluble sterol-containing metabolites such as cholesterol.

Lipids serve many functions in living organisms including nutrients,energy storage, structural components of cell membranes, and importantsignaling molecules. Although the term lipid is sometimes used as asynonym for fat, it is in fact a subgroup of lipids called triglyceridesand should not be confused with the term fatty acid.

Chemically, fatty acids can be described as long-chain monocarboxylicacids the saturated examples of which have a general structure ofCH₃(CH₂)_(n)COOH. The length of the carbon chain usually ranges from 12to 24, always with an even number of carbon atoms. When the carbon chaincontains no double bonds, it is a saturated chain. If it contains one ormore such bonds, it is unsaturated. The presence of double bonds reducesthe melting point of fatty acids. Furthermore, unsaturated fatty acidscan occur either in cis or trans geometric isomers. In a vast majorityof naturally occurring fatty acids, the double bonds are in thecis-configuration.

Polyunsaturated fatty acids (PUFA) include omega-6 (also known as ω-6 orn-6) and omega-3 (also known as ω-3 or n-3) polyunsaturated fatty acids.The designation as omega-3 or omega-6 is based on the fatty acid'sstructure, namely the distance of the first unsaturated bond from themethyl (omega) end of the fatty acid molecule. Omega-3 polyunsaturatedfatty acids mainly include cis-20:5(Δ^(5, 8, 11, 14, 17))-eicosapentaenoic acid (EPA); cis-22:5(Δ^(7, 10, 13, 16, 19))-docosapentaenoic acid (DPA); cis-22:6(Δ^(4, 7, 10, 13, 16, 19))-docosahexaenoic acid (DHA); and cis-18:3(Δ^(3, 6, 9))-α-linoleic acid. Omega-6 polyunsaturated fatty acidsmainly include cis-18:20 (^(9,12)-linoleic acid and cis-20:4(Δ^(5, 8, 11, 14))-arachidonic acid.

Glycerol is a chemical compound with the formula HOCH₂CH(OH)CH2OH.Glycerides are lipids possessing a glycerol (a crude name for which ispropan-1,2,3-triol) core structure with one or more fatty acyl groups,which are fatty acid-derived chains attached to the glycerol backbone byester linkages.

A diglyceride (“DG”), also known as a diacylglycerol, is a glycerideconsisting of two fatty acid chains covalently bonded to a glycerolmolecule tough ester linkages. A triglyceride (“TG”) (also known astriacylglycerol or triacylglyceride) is a glyceride in which theglycerol is esterified with three fatty acids. An acyl group is afunction group derived by the removal of one or more hydroxyl group andoxoacid.

Triglycerides may also be classified as having a long or medium chainlength. Long chain triglycerides preferably contain fatty acids with 14or more carbons, while medium chain triglycerides preferably containfatty acids with 6 to 12 carbons. Long chain triglycerides may includeomega-3 and omega-6 fatty acids. Medium chain triglycerides havesaturated fatty acids and thus do not contain omega-6 or omega-3 fattyacids. Long chain triglycerides (LCT) and medium chain triglycerides(MCT) may serve as energy sources. Medium chain triglycerides mayinfluence the metabolism of emulsion droplets because of their fasthydrolysis and other properties (i.e. enhancing particle binding tocells).

Lipolysis refers to the hydrolysis of a glyceride into glycerol andfatty acids. Lipolysis is the rate-determining step of lipid metabolism.The maximum metabolizing rate for exogenous long chain (e.g. for soybeanemulsion) triglycerides is about 3.8 g of lipid/kg body weight per day(Hallberg et al., Acta Physiol. Scand. (1965) Vol. 65, Suppl. 254, page16). Triglycerides typical of fish oils (i.e., triglycerides having ahigh concentration of omega-3 fatty acids e.g., higher than 30%) arehydrolyzed much more slowly than triglycerides from vegetable oils(i.e., triglycerides having predominantly fatty acids with sixteen toeighteen carbon atoms, as in soybean or olive oils), and vegetable oillong-chain triglycerides are hydrolyzed more slowly than medium chaintriglycerides (MCTs). Addition of a fish oil emulsion to a long chaintriglyceride (LCT) emulsion can even inhibit hydrolysis of long chaintriglycerides (e.g., from soybean oil) by lipoprotein lipase (LPL).

An emulsion is a thermodynamically unstable mixture of two essentiallyimmiscible liquids. Emulsions may typically be formed when twoimmiscible liquids are mechanically agitated, both phases initiallytending to form droplets. When the agitation is stopped, the dropletsquickly coalesce, and the two liquids separate. Usually, only one phasepersists in droplet form for a prolonged period of time. This phase iscalled the internal (disperse or discontinuous) phase, and it issurrounded by an external (continuous) phase. The most common types ofpharmaceutical or cosmetic emulsions include water as one of the phasesand a lipid as the other (“the lipid phase”). If the oil or lipiddroplets are dispersed in a continuous aqueous phase, the emulsion istermed oil-in-water (o/w); if the oil is the continuous phase, theemulsion is of the water-in-oil type (w/o). (Lachman et al., The Theoryand Practice of Industrial Pharmacy, 3rd Ed., 1986, p. 502.)

The lifetime of the droplets is materially increased if an emulsifier isadded to the two immiscible liquids. An emulsifier is a substance whichstabilizes an emulsion, frequently a surfactant. Examples of foodemulsifiers are egg yolk (where the main emulsifying chemical is thephospholipid lecithin), and mustard, where a variety of chemicals in themucilage surrounding the seed hull act as emulsifiers; proteins andlow-molecular weight emulsifiers are common as well. In some cases,particles can stabilize emulsions as well through a mechanism calledPickering stabilization. Both mayonnaise and hollandaise sauce areoil-in-water emulsions stabilized with egg yolk lecithin. Detergents areanother class of surfactant, and will chemically interact with both oiland water, thus stabilizing the interface between oil or water dropletsin suspension. This principle is exploited in soap to remove grease forthe purpose of cleaning. A wide variety of emulsifiers are used inpharmacy to prepare emulsions such as creams and lotions.

Emulsions have been used as a component of parenteral nutrition tosupply the body with fats in an intravenously acceptable dosage form incases where normal (oral) nutrition is impossible, compromised ormedically contraindicated or when it is necessary to promptly modify thefatty acid pattern of cells. Free fatty acids from lipid emulsions aremade available to the body either when they are hydrolytically releasedfrom the infused triglycerides via the action of lipoprotein lipase(LPL) or after cellular intake of the entire emulsion particle or itsremnants directly into cells.

Emulsions comprising triglycerides are known in the art. They includelipid emulsions prepared from vegetable oils (e.g., safflower or soybeanoils), and, in some cases, they also contain medium chain triglycerides,long chain triglycerides and/or oils of marine origin (fish oils). Suchemulsions may have a smaller weight-percentage of diglycerides and mayalso comprise omega-3 fatty acids. These emulsions comprise highpercentages of triglycerides, which are catabolized at a slower ratethan diglycerides.

Omega-3 Diglyceride Emulsions

The present invention provides a pharmaceutical composition comprising:an omega-3 diglyceride emulsion suitable for administration to apatient; and a pharmaceutically acceptable carrier, wherein the emulsionhas a lipid phase of at least about 40 wt.-% of omega-3 diglycerides,and at least about 70 wt.-% of the acyl-groups of the diglyceridescomprise EPA, DHA or a mixture thereof.

While not wishing to be bound by theory, it is believed that the omega-3fatty acid content of cell membranes of certain key organs (such as thebrain) and tissues can significantly be increased by administeringomega-3 diglyceride containing emulsions of the present invention.Moreover, omega-3 diglyceride emulsions of the present invention willlead to a much more efficient uptake of the omega-3 fatty acid to thecell membranes than triglyceride emulsions. This is the case even whenthe omega-3 diglyceride emulsions of the present invention are comparedto triglyceride emulsions which comprised of more rapidly-hydrolyzedglycerides such as medium-chain triglyceride or omega-6 triglycerides invegetable oils.

While not wishing it to be bound by theory, it is believed that theomega-3 diglyceride emulsions of the present invention are advantageousover triglyceride emulsions because diglycerides are hydrolyzed fasterthan triglycerides, and 1,2-diglycerides are also highly bioactivemolecules. Thus, omega-3 fatty acids present in the diglycerides,emulsions, are delivered to tissues rapidly while remaining (nontoxic).In addition, it is believed that diglyceride emulsion droplets are moreefficiently taken up by organs than triglyceride emulsion droplets.

Diglycerides are not produced naturally in high quantities in humans,vegetables or fish. However, methods of synthesizing diglycerides arewell known in the art, and are disclosed, for example, in U.S. Pat. Nos.2,206,168; 2,626,952; 3A10,881; 3,634,473; 3,097,098; 3,551,464;4,018,806; 5,106,542; 5,130,061; 5,142,071; 5,142,072; 5,959;128;5,434,280; 6,004,611; 6,337,414; 6,537,787; 6,749,881; and 7,081,542 allof which are incorporated herein by reference. Thus, the diglyceridesmay be obtained by trans-esterification of various oils (such as fishoil or rapeseed oil) containing omega-3 unsaturated acyl-groups, and/ormonoenoic acyl-groups with glycerol. Diglycerides may also be obtainedby esterification of a fatty acid derived from such an oil withglycerol.

Also, esterification reactions may be performed by chemical means (suchas using an alkali catalyst, i.e. sodium methoxide). Or diglycerides maybe prepared by enzymatic digestion of a triglyceride with lipase toyield a 2,3-diglyceride. The resulting 2,3-diglyceride may be furtherprocessed by isomerase to yield a 1,2- or 1,3-diglyceride. Diglyceridescomprising DHA and EPA may be isolated from reaction mixtures byconventional methods such as distillation or chromatography.

EPA and DHA may be obtained from any source. For example, EPA or DHA maybe synthetic, isolated from natural products, or obtained from fish oilby alkaline hydrolysis. Fish oil is generally the least expensive sourceof EPA and DHA. Fish oils include natural fish oils, processed fishoils, highly purified fish oil concentrates or (re-)esterified(synthetic) fish oils, including (re-)esterification of omega-3-fattyacids from cold water fish oil by triglyceride hydrolysis, purificationand concentration of the resultant omega-3-fatty acids with glycerol.Processed fish oils are described in European published patentapplication EP-A-0298293, which is incorporated herein by reference inits entirety.

Suitable exemplary fish oils include oils from cold-water fish such assalmon, sardine, mackerel, herring, anchovy, smelt and swordfish. Fishoils generally contain glycerides of fatty acids with chain lengths of12 to 22 carbons. Highly purified fish oil concentrates obtained, forexample, from sardine, salmon, herring and/or mackerel oils may have aneicosapentaenoic acid (EPA) content of from about 20 to 40 wt.-%,preferably at least about 25 wt.-%, and a docosahexaenoic acid (DHA)content of 10%, preferably at least 12%, based on the fatty acid methylesters of the fish oil concentrate as determined by gas chromatography(percent by area). U.S. Pat. No. 6,159,523, incorporated herein byreference in its entirety, discloses a method for making fish oilconcentrates. Generally, the amount of the polyunsaturated fatty acidsof the omega-6 series (such as linoleic acid) in natural fish oils islow, i.e. less than 10%, preferably less than 5%.

As used herein, “wt.-%” used in conjunction with lipid phase in anemulsion refers to “g lipid per 100 mL emulsion.” As used herein,“wt.-%” used in conjunction with diglyceride concentration refers to thepercentage of diglyceride based on the total weight of lipid.

As used herein, “wt.-%”, used in conjunction with monoglycerides ofEPA/DHA or unsaturated fatty acids EPA refers to the percentage ofmonoglycerides of EPA/DHA or unsaturated fatty acids BPA based on thetotal amount of lipid.

As used herein, “wt.-%” used in conjunction with medium chaintriglycerides refers to the percentage of medium chain triglyceridesbased on the total amount of lipid.

The present invention provides omega-3 diglyceride emulsions comprisinga total lipid phase from about 7 wt.-% to about 35 wt.-%. Preferably theomega-3 diglyceride emulsions comprise a total lipid phase from about 10wt.-% to about 20 wt.-%.

The lipid phase of the omega-3 diglyceride emulsions of the presentinvention preferably comprises from about 40 wt.-% to about 97 wt.-%diglycerides, based on the total weight of the lipid phase. Morepreferably, the lipid phase of the omega-3 diglyceride emulsions of thepresent invention comprise from about 60 wt.-% to about 97 wt.-%diglyceride based on the total weight of the lipid phase.

Based on the total amount of acyl groups, at least about 70 wt.-% of theacyl groups of the lipid phase comprise BPA, DH.A groups, or a mixturethereof. More preferably, about 75 wt.-% of the acyl groups of the lipidphase comprise EPA, DHA groups, or a mixture thereof. Most preferably,from about 75 wt.-% to about 90 wt.-% of acyl groups comprise BPA, DHAgroups, or a mixture thereof.

The molar ratio of DHA to EPA of the acyl groups of the diglycerides inthe emulsion of the present application is preferably from about 3 DHA:1EPA to about 1 DHA:1 EPA. In one embodiment, the molar ratio of DHA toEPA is about 3:1. In an alternative embodiment the DHA to EPA ratio isabout 1:1. For example, the molar ratio of DHA to EPA may be about2.5:1; about 2.0:1, about 1.5:1. In other embodiments the molar ratio ofDHA to EPA may be about 1:1.5, about 1:2 about 1:2.5, or about 1:3. Inyet another embodiment, at least about 70 wt.-% of the acyl groups ofthe diglycerides comprise either EPA or DHA only.

Omega-3 diglyceride emulsions of the present invention may furthercomprise from about 0 to about 60 wt.-% of monoglycerides of DHA and/orEPA. Preferably the monoglycerides of DHA and/or EPA are from about 0wt.-% to about 10 wt.-%, more preferably from about 0 wt.-% to about 2wt.-%, based on the total amount of lipid.

Omega-3 diglyceride emulsions of the present invention may also furthercomprise from about 0 to about 60 wt.-% free unsaturated fatty acids.Preferably the unsaturated fatty acids are from about 0 wt.-% to about 5wt.-%, more preferably from about 0 wt.-% to about 2 wt.-%, based on thetotal amount of the lipid phase.

In one preferred embodiment of the invention, omega-3 diglycerideemulsions of the present invention comprise medium chain triglycerides(MCT) and diglycerides. Such omega-3 diglyceride emulsions may containas a percent of total lipid from 0 to 90 wt.-% medium chaintriglycerides, more preferably from about 0 wt.-% to about 60 wt.-%,most preferably from about 40 wt.-% to about 60 wt.-%. The remainingpercent lipid in such emulsions preferably comprises diglycerides.Medium chain triglycerides may contain fatty acids with 6 to 12 carbons.Preferably, at least 90 wt.-% of the medium chain triglycerides aretriglycerides of caprylic acid (C₈) and capric acid (C₁₀).

Omega-3 diglyceride emulsions of the invention are preferablyoil-in-water (o/w) emulsions in which the dispersal phase compriseslipid and the outer continuous phase comprises distilled water purifiedfor parenteral purposes. Such oil-in-water emulsions may be obtained bystandard methods, i.e. by mixing the oil components followed byemulsification and sterilization. The pH value of omega-3 diglycerideemulsions of the present invention may be adjusted to a physiologicallyacceptable value, preferably to a pH of from about 6.0 to about 9.0,more preferably from about 6.5 to about 8.5. Auxiliary agents such asglycerol and additives such as antioxidants may be added to the oilmixture prior to emulsification or prior to sterilization. Omega-3diglyceride emulsions are preferably isotonic.

Methods for making emulsions are well known in the art and are describedfor example in Kirk-Othmer, Encyclopedia of Chemical Technology, 3^(rd)Ed., v. 8, pp. 900-933 (1979). See also U.S. Pat. Nos. 2,977,283;3,169,094; 4,101,673; 4,563,354; 4,784,845; 4,816,247, all of which areincorporated by reference in their entirety.

Omega-3 diglyceride emulsions according to the invention can be preparedby known standard procedures. Typically, the lipids, an emulsifier, andother auxiliary agents and additives are mixed first. Water is added tothe mixture with dispersing. The water may optionally contain additionalwater-soluble components (e.g. glycerol). Energy input through shaking,stirring, homogenizers, sonication or spray processes is typically usedto form the omega-3 diglyceride emulsion of the invention.

Emulsions thus obtained preferably contain lipid drops having a diameterof about 10 μm. Average droplet sizes of emulsions may be furtherreduced by additional homogenization, preferably by using ahigh-pressure homogenizer. Lipid droplets of the present inventionpreferably have a median particle size of less than 1 μm. For parenteralapplication, median lipid droplet sizes may be less than about 1 μm andpreferably in the range from about 100-500 nm, more preferably fromabout 100 nm to about 400 nm, most preferably from about 200 nm to about350 nm. For other applications, such as transdermal applications, meandiameter of the lipid droplet can be larger, for example, from about 1μm and 5 μm.

Method of Treatment Using Omega-3 Diglyceride Emulsions

The present invention also provides a method for preventing organ deathor injury comprising: administering to a patient a therapeuticallyeffective amount of a pharmaceutical composition which comprise anomega-3 diglyceride emulsion suitable for administration to a patient;and a pharmaceutically acceptable carrier, wherein the emulsioncomprises at least about 40 wt.-% of omega-3 diglycerides. Preferably,at least about 70 wt.-% of the acyl-groups of the diglycerides compriseof EPA, DHA or a mixture thereof.

The present invention further provides a method for reducing adversecytokine production comprising administering to a patient in needthereof a therapeutically effective amount of a pharmaceuticalcomposition comprising: an omega-3 diglyceride emulsion suitable foradministration to a patient; and a pharmaceutically acceptable carrier,wherein the emulsion comprises at least about 40 wt.-% of omega-3diglycerides. Preferably, at least about 70 wt.-% of the acyl-groups ofthe diglycerides comprise of EPA, DHA or a mixture thereof.

While not wishing to be bound by theory, it is believed that omega-3fatty acids present in the emulsion decrease pro-inflammatory cytokineproduction by suppressing production of agents (such as TNF-alpha) thatfacilitate tissue breakdown. High cytokine concentrations are believedto impair hydrolysis of circulating glycerides by lipoprotein lipase(LPL).

The present invention further provides a method for reducing cell deathor damage resulting from hypoxia-ischemia comprising administering to apatient in need thereof a therapeutically effective amount of apharmaceutical composition comprising: an omega-3 diglyceride emulsionsuitable for administration to a patient; and a pharmaceuticallyacceptable carrier, wherein the emulsion comprises at least about 40wt.-% of omega-3 diglycerides. Preferably, at least about 70 wt.-% ofthe acyl-groups of the diglycerides comprise of EPA, DHA or a mixturethereof.

Hypoxia refers to a shortage of oxygen in the body. Ischemia refers toinsufficient blood flow to provide adequate oxygenation. The most commoncauses of ischemia are acute arterial thrombus formation, chronicnarrowing (stenosis) of a supply artery that is often caused byatherosclerotic disease, and arterial vasospasm. As blood flow isreduced to an organ, oxygen extraction increases. When the tissue isunable to extract adequate oxygen, the partial pressure of oxygen withinthe tissue fails (hypoxia) leading to a reduction in mitochondrialrespiration and oxidative metabolism. Further, in many acute situationsof organ ischemia-hypoxia (e.g., stroke, myocardial infarction,intestinal volvulus, etc.) the patient is far too ill to have oral orenteral administration of therapeutic agents and thus needs parenteralinjections, such as from lipid emulsions for immediate action.

While not wishing to be bound by theory, it is believed that organ deathor injury is frequently precipitated by hypoxia-ischemia, or associatedwith hypoxia-reperfusion damage, cardiac arrhythmias, organtransplantation, endothelial dysfunction, impaired organ microperfusion, increased risk of thrombus formation, or ectopic fatdeposition, etc. Ectopic fat depositions usually occur in organs notspecialized in fat deposition, such as liver, pancreas, or heart.

The present invention also provides a method of limiting neurologicaldamage resulting from hypoxia-ischemia, comprising administering anomega-3 diglyceride lipid emulsion after a cerebral hypoxia-ischemiainsult wherein the omega-3 diglyceride lipid emulsion comprises at leastabout 40 wt.-% of a diglyceride. Preferably, at least about 70 wt.-% ofthe acyl-groups of said diglycerides comprise EPA and DHA groups.

The methods of the present invention are believed to be able to preventorgan death or injury when hypoxia-ischemia and/or its sequelae hasoccurred or is going to occur in the organs selected from the groupcomprising brain, lung, heart, kidney, spinal cord, lower or upperlimbs, and large or small intestine.

In another embodiment of the invention, methods preventing organ deathor injury, or limiting/preventing cell death and cell/tissue damageresulting from hypoxia-ischemia and/or its sequelae, compriseadministering an omega-3 diglyceride lipid emulsion of the presentinvention in conjunction with standard available therapies (such assurgery and angioplasty) and/or medications given to prevent or treathypoxia-ischemia. For example, the following drugs may be administeredwith the omega-3 diglyceride emulsion: antiplatelet medications such asaspirin, clopidogrel, dipyridamole, or ticlopidine; anticoagulants(problems of increasing hydrolysis and FFA release); and thrombolyticagents such as tissue plasminogen activator.

Omega-3 diglyceride emulsions according to the present invention may beused to treat patients with a global omega-3 fatty acid deficiency or arelative deficiency in cell membranes of certain organs or tissues,including heart, brain, kidney, lung, liver, pancreas, adipose tissue,endothelium, white blood cells, platelets and immune cells, or thosepatients with a condition benefiting from increasing availability ofomega-3 fatty acids. Emulsions and methods of the present invention maybe used for the treatment of surgical or percutaneous revascularization,such as in coronary or other “peripheral” arteries, myocardial schemiaor infarction, unstable angina, transient cerebral ischemia or stroke,inflammation, auto-immune and thrombotic diseases, such as venous orarterial diseases, organ transplantation (with infusion in both donorsand recipients), abdominal operations, multiple trauma, infections,impending or manifest sepsis, cachexia (wasting) diseases, angiographicprocedures, preterm infants (especially for raising the omega-3 fattyacid content in the brain and retina), excessive acute phase reactions,acute respiratory distress syndrome, intestinal ischemia, cardiovascularcomplications of diabetes mellitus, severe burns, Raynaud's disease,conditions of ectopic fat deposition (e.g., hepatic steatosis) andomega-3 fatty acid deficiency in cell membranes or patients unable toabsorb large amounts of omega-3 fatty acids. Omega-3 diglycerideemulsions according to the invention can also be used for administrationin patients with impaired tissue or organ perfusion, increased risk ofsevere cardiac arrhythmia (e.g. ventricular fibrillation), or duringdialysis in patients treated with haemodialysis. The invention may beadministered for pre-operative conditions or post-operative conditionsor in severe or persistent post-aggression metabolic response followingoperations. Omega-3 diglyceride emulsions of the invention may also beused in the manufacture of medicaments or pharmaceutical compositionsfor the treatment of the above-mentioned diseases.

Administration of the omega-3 diglyceride lipid-based emulsion may beeither enteral, parenteral, or transdermal. The methods ofadministration a pharmaceutical composition of omega-3 diglyceride inthe present invention may further comprise any additionaladministrations of other conventional stroke treatment or preventativemedication.

Omega-3 diglyceride lipid-based emulsions may be administered at anyeffective dose, and may be administered any time after an organ injury,or a hypoxia-ischemia insult, or operation, such as 5 to 20 minutes tosix hours after the injury, insult, or operation; or 0-12 hours afterthe injury, insult, or operation. Additional later administrations arealso contemplated, for example, an additional later administration isprovided 1-24 hours after the injury, insult, or operation. The Omega-3diglyceride lipid based emulsions may also be administered at anyeffective dose, before the injury, insult, or operation, such as 1 hourprior to the injury, insult, or operation.

Omega-3 diglyceride emulsions of the invention may contain from about 2wt.-% to about 5 wt.-% of a stabilizing or isotonizing additive, such asa polyhydric alcohol, based on the emulsion. Preferred stabilizing orisotonizing additives include glycerol, sorbitol, xylitol or glucose.Glycerol is most preferred.

In addition to distilled water, omega-3 diglyceride emulsions maycontain conventional auxiliary agents and/or additives, such asemulsifiers, emulsifying aids (co-emulsifiers), stabilizers,antioxidants, and isotonizing additives.

Emulsifiers may include physiologically acceptable emulsifiers(surfactants) such as phospholipids of animal or vegetable origin.Examples of phospholipids are egg yolk lecithin, a biologicphospholipid, a phosphatidylcholine with fixed fatty acyl chaincomposition, a glycophospholipid or a phosphatidylethanolamine.Particularly preferred are purified lecithins, especially soybeanlecithin, egg lecithin, or fractions thereof, or the correspondingphosphatides. The emulsifier content may vary from about 0.02.wt.-% toabout 2.5 wt.-%, preferably from about 0.6 wt.-% to about 1.5 wt.-% andmost preferably about 1.2 wt.-%, based on the total emulsion. In oneembodiment the emulsifier is 1.2 mg of egg yolk lecithin/100 mlemulsion.

Alkali metal salts, preferably sodium salts, of long chain, C₁₆ to C₂₈fatty acids may also be used as emulsifying aids (co-emulsifiers). Theco-emulsifiers are employed in concentrations of from about 0.005 wt.-%to about 0.1 wt.-%, preferably about 0.02 wt.-% to about 0.04 wt.-%,based on the total emulsion. Further, cholesterol or a cholesterol esteralone or in combination with other co-emulsifiers may be employed as anemulsifying aid in a concentration of from about 0.005 wt.-% to about0.1 wt.-%, preferably from about 0.02 wt.-% to about 0.04 wt.-%, basedon the emulsion.

Omega-3 diglyceride emulsions may further comprise an effective amountof an antioxidant, such as vitamin E, in particular α-tocopherol (themost active isomer of vitamin E in humans) as well as β- andγ-tocopherol, and/or ascorbyl palmitate as antioxidants and thus forprotection from peroxide formation. The total amount of alpha tocopherolmay be up to 5000 mg per liter. In a preferred embodiment the totalamount of said antioxidant is from about 10 mg to about 2000 mg, morepreferably from about 25 mg to about 1000 mg, most preferably from about100 mg to 500 mg, based on 100 g of lipid.

Omega-3 diglyceride emulsions of the invention may be administeredorally, enterally, parenterally, transdermally, intravascularly,intravenously, intramuscularly, intraperitoneally or transmucosally, andare preferably administered by intravenous injection. Thus the presentinvention also relates to a pharmaceutical composition comprisingomega-3 diglyceride emulsions as described herein, preferably forinjection into the human or animal body.

Pharmaceutical compositions of the invention may further comprisevarious pharmaceutically active ingredients. In particular, thepharmaceutically active ingredient may be delivered to a particulartissue of the body (drug targeting) in combination with emulsions of thepresent invention. Omega-3 diglyceride emulsions may include carriersfor such targeted tissue treatment. Suitable carriers may be, forexample, macromolecules linked to the emulsion droplet, or lipidmicrospheres comprising soybean oil, lecithin, or fish oil. U.S. Pub.No. 2002/0155161, incorporated herein by reference in its entirety,discloses tissue-targeted delivery of emulsions.

The pharmaceutical composition may be formulated into a solid or aliquid dosage form. Solid dosage forms include, but are not limited to,tablets, pills, powders, granules, capsules, suppositories, and thelike. Liquid dosage forms include, but are not limited to liquids,suspensions, emulsions, injection preparations (solutions andsuspensions), and the like. The choice of dosage form may depend, forexample, on the age, sex, and symptoms of the patient.

The pharmaceutical composition may optionally contain other forms ofomega-3 diglyceride emulsions and/or additional active ingredients. Theamount of omega-3 diglyceride emulsions or other active ingredientpresent in the pharmaceutical composition should be sufficient to treat,ameloriate, or reduce the target condition.

The pharmaceutically acceptable excipient may be any excipient commonlyknown to one of skill in the art to be suitable for use inpharmaceutical compositions. Pharmaceutically acceptable excipientsinclude, but are not limited to, diluents, carriers, fillers, bulkingagents, binders, disintegrants, disintegration inhibitors, absorptionaccelerators, wetting agents, lubricants, glidants, surface activeagents, flavoring agents, and the like.

Carriers for use in the pharmaceutical compositions may include, but arenot limited to, lactose, white sugar, sodium chloride, glucose, urea,starch, calcium carbonate, kaolin, crystalline cellulose, or silicicacid.

Absorption accelerators may include, but are not limited to, quaternaryammonium base, sodium laurylsulfate, and the like.

Wetting agents may include, but are not limited to, glycerin, starch,and the like. Adsorbing agents used include, but are not limited to,starch, lactose, kaolin, bentonite, colloidal silicic acid, and thelike.

In liquid pharmaceutical compositions of the present invention, theomega-3 diglyceride emulsions of the present invention and any othersolid ingredients are dissolved or suspended in a liquid carrier, suchas water, vegetable oil, alcohol, polyethylene glycol, propylene glycolor glycerin.

Liquid pharmaceutical compositions can contain emulsifying agents todisperse uniformly throughout the composition an active ingredient orother excipient that is not soluble in the liquid carrier. Emulsifyingagents that can be useful in liquid compositions of the presentinvention include, for example, gelatin, egg yolk, casein, cholesterol,acacia, tragacanth, chondrus, pectin, methyl cellulose, carbomer,cetostearyl alcohol, and cetyl alcohol.

Liquid pharmaceutical compositions of the present invention can alsocontain viscosity enhancing agents to improve the mouth-feel of theproduct and/or coat the lining of the gastrointestinal tract. Suchagents include for example acacia, alginic acid bentonite, carbomer,carboxymethylcellulose calcium or sodium, cetostearyl alcohol, methylcellulose, ethylcellulose, gelatin guar gum, hydroxyethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, maltodextrin,polyvinyl alcohol, povidone, propylene carbonate, propylene glycolalginate, sodium alginate, sodium starch glycolate, starch tragacanthand xanthan gum.

Sweetening agents such as sorbitol, saccharin, sodium saccharin,sucrose, aspartame, fructose, mannitol and invert sugar can be added toimprove the taste. Preservatives and chelating agents such as alcohol,sodium benzoate, butylated hydroxy toluene, butylated hydroxyanisole andethylenediamine tetraacetic acid can be added at safe levels to improvestorage stability.

A liquid composition according to the present invention can also containa buffer such as guconic acid, lactic acid, citric acid or acetic acid,sodium guconate, sodium lactate, sodium citrate or sodium acetate.

Selection of excipients and the amounts to use can be readily determinedby an experienced formulation scientist in view of standard proceduresand reference works known in the art.

When preparing injectable pharmaceutical compositions, solutions andsuspensions are sterilized and are preferably made isotonic to blood.Injection preparations may use carriers commonly known in the art. Forexample, carriers for injectable preparations include, but are notlimited to, water, ethyl alcohol, propylene glycol, ethoxylatedisostearyl alcohol, polyoxylated isostearyl alcohol, and fatty acidesters of polyoxyethylene sorbitan. One of ordinary skill in the art caneasily determine with little or no experimentation the amount of sodiumchloride, glucose, or glycerin necessary to make the injectablepreparation isotonic. Additional ingredients, such as dissolving agents,buffer agents, and analgesic agents may be added. If necessary, coloringagents, preservatives, perfumes, seasoning agents, sweetening agents,and other medicines may also be added to the desired preparations.

Pharmaceutical compositions of the invention may further comprisevarious pharmaceutically active ingredients. In particular, thepharmaceutically active ingredient may be delivered to a particulartissue of the body (drug targeting) in combination with micro emulsionsof the present invention. Omega-3 diglyceride emulsions may includecarriers for such targeted tissue treatment. Suitable carriers may be,for example, macromolecules linked to the emulsion droplet, or lipidmicrospheres comprising soybean oil, lecithin, or fish oil. U.S. Pub.No. 2002/0155161, incorporated herein by reference in its entirety,discloses tissue-targeted delivery of emulsions.

Omega-3 diglyceride compositions of the invention allow for rapid andefficient uptake of omega-3 fatty acids, including BPA and DHA, intocell membranes of organs and tissues. Accordingly, there is provided amethod for delivering an emulsion of omega-3 diglycerides enriched withEPA and DHA to cells and organs by administering omega-3 diglycerideemulsions of the present invention.

Lipolysis of emulsions of the invention facilitates the release of freeomega-3 fatty acids and monoglycerides into the bloodstream or in cells.Free fatty acids may be transported into mitochondria for use as anenergy source, or may be incorporated into cell membranes. Enrichingcell membranes and phospholipids with omega-3 long chain polyunsaturatedfatty acids (PUPA) may help promote or restore an adequate balancebetween omega-3 and omega-6 fatty acids. Incorporation of EPA and DHAalso increases membrane fluidity and flexibility.

The invention includes methods of treatment using omega-3 diglycerideemulsions of the present invention. The present invention also providesmeans of preventing or reducing organ lipid excess deposition and/orcell and/or organ damage/death due to hypoxia-ischemia by administeringan omega-3 diglyceride lipid emulsion of the present invention. Thus,there is generally an increase in free fatty acid concentrationfollowing an ischemic event, which inhibits mitochondrial respiratoryfunctions and leads to cell death. In the brain, for example, it isnecessary to repair cell membranes because normal membranes arenecessary for proper structure and function of synaptic membranes.

Fatty acids present in the omega-3 lipid emulsion of the presentinvention may be used as an energy source or incorporated into cellmembranes. Omega-3 diglyceride emulsions of the present invention may beused to reduce or prevent cell damage/death in any organ that may beaffected by ischemia, including but not limited to the brain, heart,kidney, lung and intestine. Preferably the emulsions are administeredeither enterally (for example, orogastric or nasogastric) orparenterally (for example, subcutaneous, intravenous, intramuscular,intraperitoneal, transdermal and transmucosal are consideredparenterally). Most preferably the emulsion is administeredintravenously.

Omega-3 diglyceride emulsions of the present invention are preferablyprovided at a dose effective for the respective treatment. For example,there is evidence that DHA and EPA are involved in cell signaling thatpromotes survival of certain cell types. N. G. Bazan, Trends Neurosci.,29(5) :263-71 (2006); Lukiw, et al., J. Clin. Invest., 115(10): 2774-83(2005); N. G. Bazan, Mol. Neurobiol., 31(1-3): 219-30 (2005). Thoseskilled in the art would be able to determine the appropriate dose basedon the experimental data presented herein. However, for example asuitable effective and tolerable dose for a human would be about 0.05g/kg to 4.0 g/kg per day, preferably about 0.3 g to about 1.5 gglyceride per kg body weight per day. Higher doses may be given asnecessary. Administration may be continuous or in the form of one orseveral doses per day. One skilled in the art would appreciateappropriate dosage and routes of administration based upon theparticular subject and condition to be treated.

Omega-3 diglyceride emulsions of the present invention are preferablyadministered parenterally and/or enterally after the ischemic insult (orin some embodiments, before the insult when it can be anticipated). Theemulsion may be administered to prevent/reduce tissue damage aftercerebral hypoxia or stroke as well as hypoxic-ischemic insults in otherorgans such as heart, kidney, lung, etc. Preferably omega-3 diglycerideemulsions of the present invention are administered as soon as possibleafter the insult (or before in cases where the insult can be predicted)to provide for a greater reduction of cell death. For example, in apreferred embodiment an omega-3 diglyceride lipid emulsion of thepresent invention is administered from 0-12 hours after the insult.Ideally the administration occurs anywhere from 20 minutes to 6 hoursafter the insult. Most preferably the emulsion is administered 0-2 hoursafter the insult. The present invention also provides for multipleadministrations of omega-3 diglyceride emulsions of the presentinvention. For example, the emulsion may be first administered before orwithin 20 minutes of the insult, followed by a second administration1-24 hours after the insult. The present invention also contemplatesmultiple administration(s) of omega-3 diglyceride emulsions followingthe insult.

The foregoing description has been directed to particular embodiments ofthe invention for the purposes of illustration and explanation. It willbe apparent to those skilled in the art, however, that modifications,changes and variations may be applied to the present invention withoutdeparting from the scope and spirit of the claimed invention. It isintended that the following claims be interpreted to embrace all suchmodifications and changes.

EXAMPLES Example 1 Method of Preparation of Diglyceride Emulsions

DG Lipid Emulsions. Phospholipid-stabilized emulsions of n-3 DG (20 g ofDG/100 ml) are prepared with fish oil DG, respectively, and egg yolkphospholipid.

Each emulsion contains 20 g of DG, which is emulsified by 1.2 g of eggyolk lecithin, and 2.5 g of glycerol/100 mL. The emulsion lipids aremixed in doubly distilled water (30 g of water and 20 g of oil) anddispersed by means of an Ultra-Turrax (Janke and Kunkel KG, Staufen,West Germany) for 10 min; water is added to give a final volume of 100mL, and emulsions are dispersed for an additional 10 min. Subsequently,the dispersion is homogenized by ultrasound in a cooling cell with aLabsonic 2000 homogenizer for 10 min at an energy input of 200 W. Theemulsions are then sealed in 5 mL vials under N2, and thereafter kept at4° C. Mean particle sizes are determined by laser spectroscopy, and bothare similar in size and homogeneity with mean diameters between 290 and300 nm. N-3 DG emulsion contains 1.42±0.25 (mean±standard deviation forthree measurements) of total DG as FFA, i.e., a range of 0.0006-0.0009mM, concentrations too low to significantly affect emulsion metabolism.

Example 2 60 Minutes of Hypoxia-Ischemia

Postnatal day 19-21 Wistar rats of both genders are subjected tounilateral (right) carotid artery ligation. See Rice, J. E., 3rd, R. C.Vannucci, et al. (1981), “The influence of immaturity onhypoxic-ischemic brain damage in the rat,” Ann Neurol 9(2): 131-41 andVannucci, S. J., L. B. Seaman, et al. (1996), “Effects ofhypoxia-ischemia on GLUT1 and GLUT3 glucose transporters in immature ratbrain,” Journal of Cerebral Blood Flow & Metabolism 16(1): 77-81.

Immediately after ligation, six rats are given 50 mg 20% diglycerideomega-3 lipid-based emulsion (0.25 cc) (a 20% long chain omega-3diglyceride-based formula having ≧70% of total omega-3 diglyceride fattyacid as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) viaorogastric feeding tube, and six control rats are given 0.25 cc water,both enterally. The 20% omega-3 diglyceride lipid-based emulsion is madeplacing 20 g of omega-3 diglyceride in 100 ml of water, and emulsifyingwith 1.2 g of egg yolk lecithin. Rats are allowed to recover for 2hours, then they undergo hypoxia-ischemia for 60 minutes of 8% oxygen ata constant temperature. The six pre-treated rats are given another doseof 50 mg omega-3 diglyceride lipid-based emulsion immediately after thehypoxia-ischemia and control rats are given another 0.25 cc water. Allrats are euthanized at 72 hours of reperfusion. The brains are removedand cut into 2 mm sections and stained with 2,3,5,Triphenyl-2H-tetrazolium chloride (TTC). TTC is a vital dye that stainscells red that have respiring mitochondria. Dead tissue (infarct)appears white.

The sections are scored as follows:

0—no evidence of edema or cell death

1—edema without cell death

2—edema with minimal cell death

3—edema with significant cell death

All rats survive 60 minutes of hypoxia-ischemia. Six of the six controlrats have edema and/or cell death with a mean score of about 2.0, whiletwo of the six treated rats are expected to have a mean score of 0.3.

Example 3 65 Minutes of Hypoxia-Ischemia

Postnatal day 19-21 Wistar rats of both genders are subjected tounilateral (right) carotid artery ligation. Immediately after ligation,six rats are given 50 mg 20% omega-3 diglyceride lipid-based emulsion(0.25 cc) (20% omega-3 diglyceride fatty acid based formula having ≧70%of total omega-3 diglyceride fatty acid as EPA and DHA) via orogastricfeeding tube and six control rats are given 0.25 cc water, bothenterally. The emulsion is made as described in Example 1. The rats areallowed to recover for two hours, and then undergo hypoxia-ischemia for65 minutes of 8% oxygen at a constant temperature. The six pre-treatedrats are given another dose of 50 mg omega-3 diglyceride lipid-basedemulsion immediately after the hypoxia-ischemia and control rats aregiven another 0.25 cc water. All rats are euthanized at 72 hours ofreperfusion. The brains are removed and cut into 2 mm sections andstained with 2,3,5, Triphenyl-2H-tetrazolium chloride (TTC).

The sections are scored as follows:

0—no evidence of edema or cell death

1—edema without cell death

2—edema with minimal cell death

3—edema with significant cell death

The 65 minutes of hypoxia-ischemia produce damage in all rats. Four ofthe six control rats survive with a mean score of around 2.5, while fiveof the six treated rats are expected to survive with a mean score of1.5.

Example 4 Treatment of Rats with Omega-3 Diglyceride Lipid EmulsionPrior to 60 Minutes of Hypoxia

Postnatal day 19-21 Wistar rats are subjected to unilateral (right)carotid artery. Immediately after ligation, six rats are given 50 mg ofa 20% omega-3 diglyceride lipid-based emulsion (0.25 cc), and sixcontrol rats are given 0.25 cc water, both enterally. The emulsion is asdescribed above in Example 1. Rats are allowed to recover for two hours,then undergo hypoxia-ischemia for 60 minutes of 8% oxygen at a constanttemperature. The six pre-treated rats are given another dose of 50 mgomega-3 diglyceride lipid emulsion immediately after thehypoxia/ischemia and control rats are given another 0.25 cc water. At 72hours of reperfusion, the rats are euthanized and their brains areremoved, cut into 2 mm sections and stained with 2,3,5triphenyl-2H-tetrazolium chloride (TTC). The damage in each animal isthen given a score from 0 (no damage) to 4 (>60% ipsilateral hemisphereinfarcted). All of the vehicle-treated animals suffer brain damage, witha mean damage score of about 2.00; the omega-3 diglyceride lipidemulsion-treated rats are expected to be significantly less damaged,having a mean damage score 0.3.

The results are expected to show that when omega-3 diglycerides areadministered immediately before and/or after hypoxia-ischemia theanimals receive significant neuroprotection. Superior results areexpected to be obtained when the omega-3 diglycerides are injectedparenterally.

Example 5 Treatment Following Hypoxia-Ischemia

Post-natal day 19-21 rat pups are subjected to unilateral carotid arterylitigation and 60 minutes of hypoxia-ischemia, according to thepreviously described protocol. On four separate occasions, rats aretreated by parenteral injection of omega-3 diglyceride lipid-basedemulsion (100 mg) immediately after the insult, and again at four hoursafter the insult. The emulsion is as described above in Example 1. Braindamage is evaluated by TTC staining at 72 hours of reperfusion. In eachinstance, administration of the omega-3 diglyceride lipid-based oilemulsion provides greater than 50% protection, i.e. reduction of tissuedamage.

The results of these experiments are expected to represent thesignificance of the overall protection. It is expected that 50% of thetreated animals are 100% protected (no damage at all, compared to 1/14untreated; 40% suffered only mild damage, compared to 1/14 mildlydamaged untreated animals). The results are expected to indicate thattreatment following hypoxia-ischemia provides a neuroprotective benefitas indicated by a reduction of tissue damage.

Example 6 Quantification of Effects of Omega-3 Diglyceride Treatment onCellular Targets

Studies on the effects of omega-3 diglyceride treatment on thegeneration of reactive oxygen species (ROS) and markers of oxidativedamage, as well as indices of inflammation at 2, 4, 8 and 24 h after thehypoxic/ischemic insult are performed. Lasting protection is expected toconfirm by brain histopathology at eight weeks following the originalhypoxia-ischemic. Sections of the brain are stained (including bothinvolved and non-involved hemispheres) with antibodies recognizingactivated proteins known to participate in neuronal apoptosis (caspase3, Jun N-terminal kinases), neuronal survival (activated Akt,phosphorylated BAD, FKHR) or to mediate the effects of NMDA-R signaling(CAM KII, and protein kinase C isoforms, in particular PKCγ and PKC

). Sections are co-stained with antibodies recognizing neuronal specificproteins (Tau), astrocytes (GFAP) or microglia. These analyses willallow quantification of the effect of omega-3 diglyceride onhypoxia-ischemia induced changes in apoptotic versus anti-apoptoticsignaling in neurons, as well as gain indices of astrocytic or microgliainvolvement. Further, whole brain extracts from involved andnon-involved hemispheres are prepared to quantify the extent of caspase,JNK and Akt activation by immunoblotting. These extracts are also usedto address the question of whether omega-3 diglycerides treatmenteffects the activation of brain sterol regulatory element bindingproteins (SREBP) in vivo.

What is claimed is: 1-22. (canceled)
 23. A method for reducing adversecytokine production comprising administering to a subject in needthereof a therapeutically effective amount of an omega-3 lipid-basedoil-in-water emulsion suitable for administration to a subject, wherein(a) the emulsion comprises at least about 7% to about 35% omega-3 oil byweight in grams per 100 ml of emulsion, (b) the omega-3 oil comprises atleast about 20% omega-3 diglyceride by weight per total weight of theomega-3 oil, and at least about 70 wt.-% of the acyl-groups of theomega-3 diglycerides comprise EPA, DHA or a mixture thereof, (c) theomega-3 oil comprises less than about 10% omega 6 fatty acids, and (d)the mean diameter of lipid droplets in the emulsion is less than about 5microns.
 24. The method of claim 23, wherein the cytokines arepro-inflammatory cytokines.
 25. The method of claim 24, wherein thepro-inflammatory cytokines comprise TNF alpha, tumor necrosis factor andinterleukins.
 26. The method of claim 23, wherein the lipid droplets areless than about 1 micron in diameter.
 27. The method of claim 23,wherein the omega-3 oil comprises from about 20% to about 40% omega-3diglyceride by weight per total weight of the omega-3 oil.
 28. Themethod of claim 23, wherein the total DHA and EPA content of the omega-3oil are present in a molar ratio of from about 3:1 to about 1:1.
 29. Themethod of claim 23, wherein at least about 75 wt.-% of the acyl-groupsof the diglycerides comprise EPA, DHA or a mixture thereof.